Process for the isolation of galanthamine

ABSTRACT

The subject matter of the present invention concerns a process for the isolation of the alkaloid galanthamine; the galanthamine itself which has been produced according to this process; the use of the galanthamine thus produced in galenic preparations; and the galanthamine thus produced for treating narrow-angle glaucoma, Alzheimer&#39;s disease, as well as alcohol and nicotine dependence.

This application is a continuation of Ser. No. 09/986,792, filed Nov. 9,2001, which is a continuation of Ser. No. 09/741,436 filed Dec. 21,2000,now U.S. Pat No. 6,335,328, which is a division of Ser. No. 09/130,883filed Aug. 7, 1998, now U.S. Pat. No. 6,194,404, which is a division ofSer. No. 08/913,461 filed Jan. 12, 1998, now U.S. Pat. No. 5,877,172,which is a 371 of PCT/EP96/01094 filed Mar. 14, 1996.

The present invention relates to a process for isolating the alkaloidgalanthamine; the galanthamine itself which has been produced by thisprocess; the use of galanthamine thus manufactured in galenicpreparations; as well as to galanthamine thus produced for the treatmentof narrow-angle glaucoma, Alzheimer's disease, as well as alcohol andnicotine dependence.

Galanthamine(4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro-(3a,3,2-ef)-(2)benzazepine-6-ol) is a tetracyclic alkaloid which, owing to itspharmacological properties, belongs to the group of reversibly actingcholinesterase inhibitors. Its effects are similar to those ofphysostigmine and neostigmine. However, it also has unique specificproperties, for example, highly analgesic effects comparable to those ofmorphine. As a cholinesterase inhibitor the therapeutic range ofgalanthamine is three to six times broader, since it is not as toxic asphysostigmine and neostigmine. This advantage compensates for itsslightly lower cholinesterase-inhibiting action, relative to dosage.Galanthamine is used in poliomyelitis and different diseases of thenervous system, but mainly in the treatment of narrow-angle glaucoma andas antidote after curare applications. The use of galanthamine in thetreatment of Alzheimer's disease is in an experimental stage. Lately,the treatment of alcohol and nicotine dependence has also been described(DE-OS 40 10 079, DE-OS 43 01 782).

The therapy of Alzheimer's disease, alcohol and nicotine dependence, aswell as that of narrow-angle glaucoma requires long-acting drug formsadapted to the particular circumstances. This could be an eye ointmentin the treatment of narrow-angle glaucoma. In the treatment ofAlzheimer's disease, alcohol or nicotine dependence, complicatedtherapeutic plans or prolonged infusions are unsuitable for obviousreasons. In these diseases, a transdermal therapeutic system (TTS) is asuitable form, for example that described in DE-OS 43 01 783. Neitherthe intact skin nor the cornea permit absorption of active substancesalts. For this reason it is not possible to use galanthaminehydrobromide or galanthamine hydrochloride with ointments or a TTS inthe therapy of narrow-angle glaucoma, Alzheimer's disease, or alcohol ornicotine dependence. The pure galanthamine base must therefore be used.

An economically efficient synthesis of galanthamine base is not possiblebecause of its complicated tetracyclic structure with three opticallyactive carbon atoms. For this reason, galanthamine is usually isolatedfrom plants belonging to Amaryllidaceae, for example, from galanthusspecies, such as the snowdrop or Leucojum aestivum. These plants havethe advantage of comprising galanthamine in concentrations of up to 0.3%with only small amounts of companion alkaloids so that the extractionmethod described in DE-PS 11 93 061 can be used. However, both thegalanthus species and Leucojum aestivum are protected. On the otherhand, the extraction method described in DE-PS 11 93 061 preferably useschlorohydrocarbons which have fallen into discredit for toxicologicalreasons. The Pharmacopoeias of the Western World therefore call for alimitation of the residual chlorohydrocarbon content to <10 ppm. In thepreparation of drugs chlorohydrocarbons should therefore be avoided asfar as possible. Moreover, in the known process, the solvent extractmust be adsorbed to alumina to ensure separation of the resinoussubstances and companion alkaloids. From the solution obtained afterfiltering off the alumina, the galanthamine is then purified bygalanthamine hydrobromide; this involves disposal of halogen salts. Forthe use in ointments or in a TTS, the galanthamine base must thenadditionally be liberated from this galanthamine hydrobromide.

It is accordingly the object of the present invention to provide aprocess for the isolation and purification of galanthainine, which doesnot have the drawbacks of the prior art processes. In particular,purification is to be facilitated, the use of chlorohydrocarbons andpurification by means of galanthamine salts are to be avoided. Theobject is achieved according to the present invention by a processhaving the characterizing features as described herein. Preferredembodiments are further disclosed.

In detail, the subject matter of the present invention is a process forthe isolation of galanthamine from biological material which isrecovered from agriculturally cultivated amaryllidacea species or fromthose which are commonly regarded as “weeds” and are not protected,preferably from the bulbs of these plants. These amaryllidaceae include,for example, narcissi or crinum species. Particularly suitable areNarcissus pseudonarcissus “Carlton” or the Asian climber Crinum amabile.Although these plants only have a tenth of the galanthamine amountcontained in the protected plants, and, moreover, have up to twelvecompanion alkaloids, the process according to the present inventionsurprisingly makes it possible to isolate galanthamine base therefromwith a purity suitable for the use in drugs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an HPLC-chromatogram of the oily residue obtained in Example2.

FIG. 2 is an HPLC-chromatogram of the galanthamine product of Example 3.

According to the present invention galanthamine-containing, biologicalmaterial, which is preferably comminuted and mixed with alkali powder,preferably with sodium hydroxide pellets, soda, potash, or similar saltssuitable to liberate bases from biological material and to preparepharmacological active substances, is extracted with toxicologicallysafe organic solvents, and the galanthamine from this extract ispurified by means of liquid-liquid extraction. It is particularlyimportant for the success of the process according to the presentinvention to maintain the pH-value in the first stage of theliquid-liquid extraction. The liquid-liquid extraction is carried out ina first stage at a pH of about 4, and in a second stage at a pH of about9. In addition to concentrated ammonia, soda solution of another basemay be used to adjust the pH-value. Diethyl ether or specialboiling-point gasoline is used as toxicologically safe organic solvent.Special boiling-point gasoline is preferably used, with it a certainpreliminary purification can be achieved. The solvent is removed andgalanthamine is recrystallized from a suitable solvent, preferably fromisopropanol. The white galanthamine base having a melting point of 129to 130 ° C. is obtained. The purity of the galanthamine thus obtained isshown in the HPLC-chromatogram (FIG. 2). The purity of the galanthamineis ≧99%.

This result is very amazing since the process according to DE-PS 11 93061 used so far was found to be unsuitable to isolate galanthamine frombiological material containing small amounts of galanthamine and up to12 companion alkaloids. The process described in DE-PS 11 93 061 resultsin an emulsion which cannot be broken so that an extraction is renderedimpossible. A slightly modified process resulted in an oily residuewhich, according to HPLC-chromatogram (FIG. 1), is contaminated by atleast four substances.

It must therefore be regarded as a particular surprise that the freegalanthamine base cane be isolated in pure form and good yield frombiological material comprising small amounts of galanthamine in additionto a large number of companion alkaloids by means of the simple processaccording to the present invention, without having to carry out anadditional adsorption step to aluminum oxide.

The galanthamine base produced according to the present invention may beused for the treatment of narrow-angle glaucoma, Alzheimer's disease, oralcohol and nicotine dependence. In addition, the galanthamine baseproduced according to the present invention may be used in galenicpreparations, such as ophthalmic ointments or transdermal therapeuticsystems, which may also be used to treat the above-mentioned diseases.

The present invention is more particularly described in the examplesthat follow, which are intended to illustrate, but not to limit, thepresent invention:

EXAMPLE 1 Comparison

In accordance with the process described in DE-PS 11 93 061, 10 kgair-dried, comminuted bulbs of Narcissus pseudonarcissus “Carlton” iscarefully mixed with 2.5 l of a 8% aqueous ammonia solution. Thematerial swells; the whole batch pastes up. The addition of 23 ldichloroethane intended for extraction results in an emulsion whichcannot be broken.

EXAMPLE 2 Comparison

The process for the isolation of galanthamine known in the art (DE-PS 1193 061) is modified. 10 kg air-dried, comminuted bulbs of Narcissuspseudonarcissus “Carlton” is carefully mixed with 400 g sodiumcarbonate. 23 l dichloroethane is added. The mixture is allowed to standfor 10 hours; then the solvent is decanted. The bulbs are once againdoused with 23 l dichloroethane which is decanted after 2 to 3 hours.After that, 17 l dichloroethane is added to the bulbs for the thirdtime; however, this is decanted immediately. The mixed dichloroethaneextracts are extracted by means of 10% sulfuric acid (2×600 ml each;2×300 ml each). The acidic extracts are mixed and purified from tracesof dichloroethane by means of shaking out with diethyl ether. Understirring and cooling to 15 to 20° C., about 200 ml of a 25% aqueousammonia solution is then added up to alkaline litmus reaction. The pH isin the range of 7 to 8. Different from the indications in the art, thecompanion alkaloids do not precipitate. The alkaline solution issaturated with salt and extracted with diethyl ether. After evaporationof the ether, a negligible residue remains, which is also different fromthe indications in the art. The pH-value of the aqueous phase is set toabout 14 by saturating it with potash. The aqueous phase is repeatedlyextracted with diethyl ether. The mixed ether extracts are evaporated todryness, the remaining galanthamine-containing residue is dissolved inacetone (50 ml). In contrast to the art, there is no precipitate. 350 mlacetone is replenished, 200 g aluminum oxide is added, and stirring iseffected for 45 minutes. The aluminum oxide is filtered off and washedtwice with 100 ml acetone each time. The mixed acetone solutions areevaporated to dryness. 1.3 g of an oily residue is obtained which isexamined by means of HPLC. The chromatogram is shown in FIG. 1. The mainpeak, the galanthamine, is marked. It is clearly visible that thegalanthamine thus isolated is contaminated by at least four substances.

EXAMPLE 3

100 kg air-dried, comminuted bulbs of Narcissus pseudonarcissus“Carlton” is carefully mixed with 4 kg of sodium carbonate. The mixtureis divided into three equal parts, and each is doused with 15 l specialboiling-point gasoline 80/110. The mixtures are allowed to stand for 24hours. The solvents are each renewed twice, collected, and evaporated todryness in low vacuum. The extracts are placed in 2% aqueous sulfuricacid and adjusted to a pH of 4 with concentrated aqueous ammoniasolution. Five extractions with diethyl ether follow. The aqueous phaseis set to a pH of 9 with concentrated ammonia and extracted five timeswith diethyl ether. These ether fractions are collected, dried withsodium sulfate, and evaporated. 20 g of a slightly yellow, oily residueis obtained, which is recrystallized from hot isopropanol. 10 g of whitegalanthamine base having a melting point of 129-130° C. is obtained.Only one peak is visible in the HPLC chromatogram (FIG. 2).

What is claimed is:
 1. A method for purification of galanthamine, whichcomprises recrystallizing a galanthamine-containing residue with asuitable solvent having a residual chlorohydrocarbon content of lessthan 10 ppm.
 2. A method according to claim 1, wherein said suitablesolvent is a toxicologically safe organic solvent.
 3. A method accordingto claim 1, wherein said suitable solvent comprises a member selectedfrom the group consisting of diethyl ether, isopropanol, and specialboiling-point gasoline 80/110.
 4. A method according to claim 1, whereinthe galanthamine obtained by said method has a purity of ≧99%.
 5. Amethod according to claim 1, wherein said galanthamine-containingresidue is a mixture selected from galanthamine-containing biologicalmaterial or synthetic galanthamine-containing material.
 6. A methodaccording to claim 1, wherein said galanthamine-containing residue is agalanthamine-containing salt which is suitable to liberate a base.
 7. Amethod according to claim 1, wherein said galanthamine-containingresidue is galanthamine hydrobromide.